Evaluation of the spot urine sampling technique
to assess urinary pseudouridine excretion

in lactating dairy cows
Kevin J. Shingfield

Grassland and Ruminant Science Department, SAC Auchincruive, Ayr, KA6 SHW, United Kingdom , Current
address: Agricultural Research Centre ofFinland, Animal Production Research, FIN-31600 Jokioinen,

Finland, e-mail: kevin.shingfield@mtt.fi
Nicholas. W. Offer

Grasslandand Ruminant Science Department, SAC Auchincruive, Ayr, KA6 SHW, United Kingdom

The potential of the spot urine sampling technique to assess urinary pseudouridine excretion was
evaluated. Twelve multiparous Holstein-Friesian cows were fed two experimental diets in a complete
change-over design with two 14 day experimental periods. Diets were either silage fed ad libitum
with a concentratesupplement offered as a single meal (SF), or a complete diet formulated from the
same ingredients (CD). Total urine collections were performed for 24h at 2 h intervals on days 11 and
14. Pseudouridine and creatinine excretion during each 2h interval depended on time of collection

(pseudouridine, P<o.ool and creatinine, P<0.05) and on cow (pseudouridine, P = 0.092 and creati-
nine, PcO.Ol), but were unaffected by differences due to sampling day or treatment. Variations in the
molar ratio of pseudouridine to creatinine(Ps/c) followed similar diurnal patterns observed for pseu-
douridine excretion. Data was used to assess the accuracy of spot urine sampling to predict daily
mean Ps/c ratios. Collection of multiple samples within a day was more reliable than collecting fewer
samples over several days, while prediction errors were generally greater for CD compared to SF.
Even the most intensive sampling regimen did not allow an acceptable prediction of the daily mean
Ps/c ratio, minimum r values 0.528 and 0.080 for SF and CD treatments, respectively. Furthermore,
mean Ps/c ratios accounted foronly half of the variation observed in daily pseudouridine excretion, A
total urine collection appears necessary to assess accurately daily pseudouridine excretion in dairy
cows.

Key words: creatinine, dairy cows, pseudouridine, spot sampling

ntroduction duction, while body protein content may also
decrease (Gibb et al. 1992), the extent of which
may be greater during protein deficiency (Bolts
et al. 1979). The importance of labile protein
reserves on subsequent lactationperformance has

During early lactation, body fat reserves are
mobilised in the dairy cow to support milk pro-

© Agricultural and Food Science in Finland
Manuscript received November 1997

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Shingfield, K.J. & Offer, N.W. Assessment ofpseudouridine excretion by spot urine sampling

recently been demonstrated in dairy cows (Moor-
by et al. 1996). Condition scoring is often used
to estimate body fat content but is of little value
as an indicator ofnitrogen status since it accounts
only poorly for variations in carcass composi-
tion.

Pseudouridine is one of the most important
modified pyrimidine derivative constituents of
both transfer and ribosomal RNA (Sander et al.
1986a). Pseudouridine liberated during tissue
RNA degradation is not salvaged, but obligately
excreted in the urine (Weissman et al. 1962).
Schöch et al. (1982) suggested that urinary pseu-
douridine excretion reflects RNA turnover and
could therefore be used as a marker of protein
synthesis (Sander et al. 1986b), assuming these
processes are directly related. In cattle, pseudou-
ridine excreted in urine is positively correlated
with growth rate in young animals, and could
potentially be used as an index of nitrogen sta-
tus (Puchala et al. 1993). Urinary creatinine ex-
cretion has been proposed as an internal marker
of urinary output (e.g. Erb et al. 1977), and there-
fore the possibility exists of assessing pseudou-
ridine excretion from the molar ratio of pseu-
douridine to creatinine (Ps/c) of spot urine sam-
ples. The current study was conducted to evalu-
ate the accuracy of the spot urine sampling tech-
nique to assess urinary pseudouridine excretion.
Data from this study used to evaluate the accu-
racy of this approach to assess urinary purine
derivative excretion has previously been report-
ed (Shingfield and Offer 1998).

Material and methods

Animals and management
Twelve multiparous lactating Holstein-Friesian
cows ofmean live weight 611 kg (s.e. 19.5) were
housed in individual stalls within the dairy cow
metabolism unit at SAC, Auchincruive. Cows
were retained in each stall with de Boer yokes.
Each stall was fitted with a rubber mat and bed-

ded with sawdust. Animals had continual access
to water and were milked in situ at approximately
0700 and 1500.

Experimental diets
Since the spot sampling technique wouldbe ap-
plied in practice over a range of diets and feed-
ing strategies, experimental diets were formu-
lated to examine the effect of feeding frequency
on the accuracy of spot urine sampling. Differ-
ences in feeding frequency were achieved by
feeding silage and a standard concentrate either
separately (treatment SF) or as a complete diet
(treatment CD). Diets were formulated from a
first cut silage ensiled during may 1992 from
grass swards in which perennial ryegrass pre-
dominated, supplemented with 7 kg fresh weight /d
of a standard concentrate to meet metabolisable
energy (ME) and metabolisable protein require-
ments (AFRC 1992) using SAC advisory ration-
ing software (N.W. Offer, pers. comm.). The
same software was used to predict ad libitum
silage intake allowing treatment CD to be for-
mulated to the same forage concentrate ratio
(62:38, on a dry matter basis) as for SF. CD was
prepared in 600 kg fresh weight batches on a
daily basis using a Keenan (Richard Keenan
Company Limited, Co. Carlow, Eire) diet feed-
er. Fresh silage and CD were offered at 0800 and
topped up as and when necessary to ensure 10%
refusals. Concentrate was offered at 1000 and
was consumed rapidly, typically within 90 min-
utes.

Experimental design
Experimental treatments were evaluated using a
complete change-over design. Cows were
grouped into two blocks of six animals, accord-
ing to calving date, parity and live weight. Mean
days in lactation, live weight (kg) and parity were
148, 610 (s.e. 22.3), 4.3; and 148, 613 (s.e. 34.4)
and 3.8, for blocks 1 and 2, respectively. Each
experimental period lasted for 14 days with

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AGRICULTURAL AND FOOD SCIENCE IN FINLAND



measurements performed on days 11 and 14.Use
of a 10 day rumen adaptation period was con-
sidered adequate since all experimental animals
had previously been fed the same silage and con-
centrate.

Measurements and sampling
Milking and feed sampling protocols have pre-
viously been reported (Shingfield and Offer
1998). Cows were weighed at the beginning and

end of each 14 day experimental period at 1000.
On days 11 and 14 of each experimental period,
2h urine collections were performed over a 24h
period, starting at 0800. Urine collection was
achieved by inducing cows to urinate by vulval
stimulation. This procedure was repeated 3 min
later to minimise end of collection errors. The
volume of urine voided for each 2h collection
was measured, whilesub-samples were taken and
stored at -20°C. Cows were observed constant-
ly to ensure a total urine collection for each an-
imal was achieved.

Chemical analyses
Chemical composition (organic matter, crude
protein, starch, neutral cellulase and gamminase
digestibility and acid hydrolysis ether extract of
feeds was determined using standard procedures
describedby Dewhurst et al. (1996). Concentrate
ME content was calculated from neutral cellula-
se and gamminase digestibility and acid hydrol-
ysis ether extract measurements according to
equation E 3 (Thomas et al. 1988). Silage in vit-
ro organic matter digestibility measurements
made using a modified (Alexander 1969) Tilley
and Terry (1963) method were used to predict
silage ME (MAFF 1975). Urinary pseudourid-
ine and creatinine concentrations were deter-
mined by High Perfomance Liquid Chromatog-
raphy (Shingfield and Offer 1998). Separation
was achieved using a Spherisorb spm ODS II
C- 18 reversed-phase column (250 mm x 4.6 mm
i,d,; Phase Separations Ltd, Deeside, Clwyd)

eluted with a mobile phase (7.5 mM phosphate
buffer, 10 mM sodium 1-heptanesulphonic acid,
1.0 mM triethylamine adjusted to pH 3.0 using
10% (v/v) hydrochloric acid) at a flow rate of
1.0 ml/min and temperature 20°C, while peaks
were detected at 218 nm. Average recovery of
pseudouridine and creatinine standards added to
urine was above 90% while within and between-
day variability was less than 4%.

Statistical analysis
Statistical analysis of all experimental data was
performed using GENSTAT 5 (version 5.3.,
Lawes Agricultural Trust, 1987). With experi-
mental data collected at regular intervals within
a period of time, the problem arises of depend-
ence ofa measurement on previous observations.
Two-hourly pseudouridine and creatinine (both
excretion and concentration) and the molarratio
of pseudouridine to creatinine (Ps/c) data was
tested for dependence, using “antorder” anttest
procedures (Gabriel 1962,Kenward 1987) which
fit data to a model which takes dependence into
account. Results of the “antorder” procedures
showed that there was no evidence to support
the fitting of a repeated measures model to the
data. Consequently, a split-split-plot analysis of
variance was used to test the effects of cow and
diet (whole-plot factors), sampling day (sub-plot
factor), sampling hour (sub-sub-plot factor) and
their interactions using the following model:-
{blocking structure = (cow x period/day/hr) and
treatment structure = (cow x day) + (diet x hr)}.

The accuracy of spot urine sampling meth-
ods were assessed using daily mean Ps/c ratios
obtained by total urine collection (i.e. sum of all
2h collections) for each cow as reference val-
ues. Initially, Ps/c ratios determined from spot
samples at each 2h sampling (n = 12) were com-
pared with reference values for each group of
experimental animals for each period (i.e. 12
measurements) producing 52 correlations. How-
ever, in practice more than one spot sample
would be collected to assess the daily mean.
Consequently, spot sampling regimens based on

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Shingfield, K.J. & Offer, N.W. Assessment ofpseudouridine excretion by spot urine sampling

Table I. Chemical composition of silage, concentrate and complete diet (g/kg dry matter (DM) unless
otherwise stated).

Silage Complete diet Concentrate

Dry matter (g/kg Fresh weight) 298 363 876
Corrected dry matter (DM; g/kg FW) 321 (l) ND ND
pH 3.9 4.2 ND
Ammonia-nitrogen (g/kg total N) 103 92.5 ND
Composition of DM
Crude protein 180 180 207
Ash 83 81 90
Neutral cellulase and gammanase digestibility ND ND 782
In vitro organic matter digestibility (g/kg OM) 764 ND ND
Acid hydrolysed ether extract 53.3 65.6 95.7
Metabolisable energy (MJ/kg DM) 11.6 (2) 11.8 <3) 13.4 l4)

(l> Dry matter corrected for loss of volatiles (Shingfield and Offer 1998)
121 calculated according to MAFF (1975)
<3> calculated using DM and ME values of silage and concentrate
(4> calculated according to the equation ofThomas et al. (1988)
ND = Not determined

2 samples collected at 12 h intervals (12 h
scheme), 3 samples collected at 8 h intervals
(8 h scheme) and 4 samples collected at 4 h in-
tervals (4 h scheme) were evaluated. Sampling
regimen Ps/c estimates were derived using all
data collected during the 24h collection period
and were correlated with the measured (by total
urine collection) daily mean Ps/c ratio, for each
cow within a group for both sampling days within
a period. To assess the increase in accuracy from
using two sampling days, the process described
above was repeated using data from both sam-
pling days. Exploratory data handling and regres-
sion analysis were undertaken using MINITAB
statistical software (Minitab Inc. 1980).

Results

Animal production
Chemical composition ofsilage, concentrate and
complete diet is shown in Table 1. Mean treat-
ment effects on dry matter, crude protein and ME
intake or milk yield and composition were not

significant (P>0.05; Table 2). Mean cow live
weights (kg) were 615 and 617 for treatments
SF and CD respectively (P>0.05).

Urinary pseudouridine concentration
and excretion

The effect of dietary treatment on daily mean
urinary pseudouridine concentration of 437 and
439 (s.e.d. 14.9) |imol/l, for SF and CD treat-
ments, respectively or daily excretion of 8.06 and
8.33 (s.e.d. 0.33) mmoles/day, for SF and CD
treatments, respectively were not significant
(P values of 0.909 and 0.431, respectively). Two-
hourly pseudouridine concentration was signifi-
cantly affected by cow (PcO.001), sampling time
(P<0.001) and by treatment x sampling time
interactions (P<0.05), Mean (n = 24) 2h pseu-
douridine concentration varied diurnally between
346-561 and 344-548 pmol/1 for SF and CD
treatments, respectively.

Two-hourly pseudouridine excretion was sig-
nificantly different between sampling times
(PcO.001) and by interactions between treatment
and sampling times (P<0.05), while differences
due to cow approached significance (P<0.092).

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Variations in mean pseudouridine excretion be-
tween each 2h sampling interval followed a di-
urnal pattern, the extent of which was greater
for SF (0.46 to 0.91 mmoles) than CD (0.52 to
0.87 mmoles) treatments (Figure 1). Daily mean
pseudouridine excretion was positively correlat-

ed with daily mean pseudouridine concentration
(r = 0.500, n = 48, P<0.05) and metabolic live
weight (r = 0.299, n = 48, Pc0.05). Coefficients
of variation (CV) due to the effects of cow, ex-
perimental period, sampling day and sampling
time on urinary pseudouridine concentration and
excretion are shown in Table 3.

Table 2. Effect of dietary treatment on nutrient intake, milk production and urinary output.

Daily intake Separate feeding Complete diet SED P

Total dry matterintake (kg) 20.1 19.8 0.557 >0.05
Crude protein (g) 3558 3569 98.5 > 0.05
Metabolisable energy (MJ) 245 234 7.1 >0.05
Milk yield and composition
Milk yield (kg) 28.8 27.5 0.61 >0.05
Fat (g/kg) 39.7 37.0 1.76 >0.05
Protein (g/kg) 30.8 31.0 0.22 >0.05
Lactose (g/kg) 47.4 46.4 2.17 >0.05
Fat (g/d) 1149 1037 90.7 >0.05
Protein (g/d) 889 850 56.7 >0.05
Lactose (g/d) 1361 1275 87.0 >0.05
Urinary output (1/d) 19.1 19.9 0.36 >0.05

Fig. 1.Diurnal variation in urinary
pseudouridine excretion for cows
offered concentrate separately
(•—) or as a complete diet
(O—). Each point is the mean
of 24 observations with s.e. Lines
without markers indicate treat-
ment daily means.

349



Shingfield, K.J. & Offer, N.W. Assessment ofpseudouridine excretion by spot urine sampling

Table 3. Coefficients of variation (CV%) forconcentration and daily excretion ofpseudouridine and creat-
inine and Ps/c ratios.

Source of variation
Cow Sampling period Sampling day Sampling hour

Concentration
Pseudouridine 16.8 8.1 18.5 41.1
Creatinine 18.3 6.4 13.0 34.5

Excretion
Pseudouridine 10.5 9,1 17,0 41.6
Creatinine 15.4 7,8 9,1 32.3

Ratio
Ps/c 10.8 6.5 10.8 27.3

Diurnal variation in urinary Ps/c ratio
Urinary creatinine concentrations reported pre-
viously (Shingfield and Offer 1998) were used
to calculate Ps/c ratios. Use of creatinine as an
internal marker of urinary output reduced varia-
tions in pseudouridine concentration between
sampling days and sampling intervals (Table 3).

Daily mean Ps/c ratios were not significant-
ly differentbetween treatments (P = 0.785, mean
0.062 and 0.063 (s.e.d. 0.002) for SF and CD
treatments, respectively) or sampling days
(P = 0.171, mean 0.061 and 0.064 (s.e.d. 0.002)
for days 1 and 2, respectively). In contrast, dai-
ly mean Ps/c ratios were significantly different
between cows (P<0.01) and were negatively cor-
related with metabolic live weight (r= - 0.59, n
= 48, P<0.001). Mean two-hourly Ps/c ratios
were significantly influenced by sampling time
\ treatment interactions (P<0.05) and were sig-
nificantly different between sampling times
(P<0.00l), ranging between 0.051 to 0.091 and
0.054 to 0.076 for SF and CD treatments, respec-
tively (Figure 2).

Accuracy of spot sampling for assessing
daily Ps/c ratios

Accuracy of the spot sampling technique was
initially assessed by comparing estimates of Ps/c

ratios from individual spot samples collected
at each sampling interval with the daily mean
obtained by total urine collection. Correlations
between daily mean Ps/c ratios and estimates
based on the collection of single spot samples
(Figure 3) were generally poor (mean r value
0.558), while differences due to treatment were
inconsistent between sampling intervals (mean
r values 0.593 and 0.523 for treatments SF and
CD, respectively).

In practice however, more than one spot sam-
ple would be used to assess daily mean Ps/c ra-
tios. Consequently threemultiple sampling meth-
ods were evaluated. Mean Ps/c ratios were de-
rived from the appropriate spot samples for each
sampling regimen and compared to the daily
mean obtained by total urine collection (Table
4). This process was repeated using estimates
based on spot samples obtained over two sam-
pling days to investigate whetheraccuracy could
be improved by extending the observation peri-
od. Collection of multiple spot samples within a
day improved correlations between spot sample
estimates and the reference mean, while only
minor improvements in spot sampling accuracy
were achieved by collecting urine over two days.
The most intensive sampling regimen (4-h sam-
pling) gave average correlations (across both SF
and CD treatments) of 0.824 and 0.743 based on
single and two day urine collections, respective-
ly, while respective minimum values were 0.679
and 0.075.

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351

Fig. 2. Diurnal variation in the mo-
lar ratio of urinary pseudouridine
to creatinine forcows offered con-
centrate separately (•—) or as
a complete diet (O—). Each
point is the mean of 24 observa-
tions with s.e. Lines without mark-
ers indicate treatmentdaily means.

Fig. 3. Correlation coefficients
between Ps/c ratio in spot urine
samples taken at different times
and the mean daily Ps/c measured
by total collection (n=l2; mean of
two correlations, range shown as
error bars).

Vol. 7(1998): 345-356.



Shingfield, K.J. cfc Offer, N.W. Assessment ofpseudouridine excretion by spot urine sampling

Table 4. A summary of correlation coefficients derived between sampling regimen estimates and daily
mean Ps/c ratios.

Sampling Number of Number of Separate feeding Complete diet feeding
regimen (hours) samplingdays correlations Min Max Mean Min Max Mean

12 I 14 1 0.348 0.922 0.615 0.506 0.837 0.618
8 I 10' 0.602 0.957 0.798 0.579 0.903 0.740
4 1 14 1 0.708 0.931 0.848 0.679 0.896 0,800
12 2 142 0.357 0.953 0.744 0.185 0.988 0.575
8 2 102 0.648 0.981 0.869 0.075 0.967 0.654
4 2 142 0.528 0.986 0.839 0.080 0.946 0.647

1 Correlations were derived using 12 units of data, obtained from 6 cows for both sampling days within
each period
2 Correlations were derived using 6 units of data, obtained from the mean of both sampling days within
each period for each cow

Mean r values based on a single sampling day
were relatively similar for both treatments (Ta-
ble 4), while mean r values based on two-day
urine collections were higher for treatment SF.
Minimum r values were higher for treatment SF
for every sampling method tested, except for 12
h sampling based on a single day urine collec-
tion.

Relationship between urinary pseudourid-
ine excretion and Ps/c ratios

Daily mean Ps/c ratios obtained by total urine
collection were poorly correlated with daily
pseudouridine excretion (r = 0.360, n = 48,
P<0.05; Table 5). The most accurate prediction

of pseudouridine excretion was attained by
weighting daily mean Ps/c data by daily urinary
creatinine excretion for each individual cow.
Adopting this approach is however impractical
since assessment of urinary creatinineexcretion
requires a total urine collection. Weighting dai-
ly mean Ps/c ratios of individual cows by uri-
nary creatinine concentration, live weight or
metabolic live weight improved the prediction
of urinary pseudouridine excretion. Use of live
weight as a weighting factor gave a relationship
which accounted for approximately half the var-
iation in pseudouridine excretion.

Discussion
Table 5. Relationships between scaled daily mean molar
ratios of pseudouridine tocreatinine (Ps/c) with pseudouri-
dine excretion.

Since, milk production, urinary purine deriva-
tive and creatinine excretion data has been dis-
cussed previously (Shingfield and Offer 1998),
discussion is confined to examination of the
sources of variation of urinary psuedouridine
excretion and evaluation of the accuracy of the
spot sampling technique to assess it.

Weighting
factor

Ps/c
Pr

None 0.36 <0.05
0,62 <O,OOl
0.56 <O.OOl
0.62 <O.OOl
0.70 <0.001

Creatinine concentration
Metabolic live weight
Live weight

Urinary pseudouridine excretionLive weight squared
Live weight and creatinine
concentration 0.40 <0.01

0.98 <0.001
Urinary pseudouridine excretion varied diurnal-
ly, the extent of which was similar for both sep-Creatinine excretion

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AGRICULTURAL AND FOOD SCIENCE IN FINLAND



arate and complete diet feeding. Chen et al.
(1992) adopted a similar approach used in the
current study and attributed observed diurnal
variations in urinary purine derivative excretion
to end-of-urine collection errors. End-of-urine
collection errors were minimised in the current
study by inducing cows to urinate twice at each
collection interval. Furthermore, bias which may
be introduced as a result of systematic analyti-
cal error was minimised by analysing samples
in random sequence.

Pseudouridine derived during RNA turnover
is not salvaged or further metabolised (Borek et
al. 1977, Gehrke et al. 1979) but excreted in
urine. Since its excretion is independent of lev-
els contained in the diet (Weissman et al. 1962)
or duodenal nucleic acid flow (Puchala et al.
1993), observed variations in urinary pseudou-

ridine excretion may reflect diurnal changes in
tissue RNA turnover rates.

Daily urinary pseudouridine losses of 65.6
(s.e. 1.9) and 67.7 (s.e. 2.6) pmol/ kg W07

5, (SF
and CD treatments, respectively) were double
those reported by Puchala et al. (1993) for heif-
ers (30.8 to 36.4 pmol/ kg W 0 75) and three times
higher than values for non-lactating cows (19.5
to 21.6 pmol/ kg W07 5). Discrepancies between
these findings may potentially be explained by
metabolic processes occurring during lactation
rather than due to secretion in milk per se, since
milk protein synthesis is estimated to account
for at least 20% of whole-body synthesis in a
cow yielding 22 kg milk/d (Oldham et al. 1980)
and pseudouridine secretion in milk is thought
to be closely related to protein synthesis in the
mammary gland (Rosskopf et al. 1991). Based
on assumed milk pseudouridine concentrations
of 4 pmol/1 (Tiemeyer et al, 1984, Rosskopf et
al. 1991), daily mammary secretion may have
currently accounted for pseudouridine losses
approaching 1.0 pmol/ kg W07 5.

Other studies have demonstrated that urinary
pseudouridine excretion increases during late
pregnancy (Martin Orue et al. 1995) and growth
(Puchala et al. 1993) both of which were attrib-
uted to elevated tissue turnover of RNA and
tRNA (the major source of pseudouridine) in

particular, associated with a higher rate of pro-
tein synthesis. Comparison ofdaily urinary pseu-
douridine excretion reported for lactating (12.9
pmol/ kg W 075

, Martin Orue et al. 1996) and
non-lactating (20.4 pmol/ kg W ° 75

, Puchala et
al. 1993) adult ewes suggest that physiological
factors other than lactation per se may also be
involved.

Changes in body composition of dairy cows
during the first 29 weeks of lactation indicate
that daily net losses of crude protein approach
0.1 kg during the first eight weeks post partum
(Gibb et al. 1992). Despite a relatively large de-
pletion of crude protein from the carcass (0.123
kg/d) and smaller net losses from the udder and
urino-genital tract (0.019 kg/d) during this peri-
od, protein was accreted in the liver and diges-
tive tract (0.006 and 0.010 kg/d, respectively).
Examination of changes in body composition
indicated that throughout the first 29 weeks of
lactation, protein was accreted in the liver, gut,
heart, respiratory tract and spleen, tissues which
have a high fractional rate of protein synthesis.
Assuming that pseudouridine distribution and
turnover is similar between and within mamma-
lian species (Kahn et al. 1978), and that its ex-
cretion reflects RNA turnover (Schöch et al.
1982) and consequently protein synthesis (San-
der et al. 1986b), changes in body composition
occurring during lactation may account for dis-
crepancies in urinary pseudouridine excretion
between animals in different physiological states.

Diurnal variation in the Ps/c ratio
Use of creatinine as an internal marker of uri-
nary output reduced variations in pseudouridine
concentrations between sampling times which is
consistent with other studies (Chen et al. 1992,
Gonda and Lindberg 1994). Within-day varia-
tions in urinary Ps/c ratios followed diurnal pat-
terns for both experimental diets, the extent of
variation being greater for separate feeding. This
finding suggests that limitations in the number
of spot samples that could be collected in prac-
tice would lead to difficulties in establishing a

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Shingfield, K.J. & Offer, N.W. Assessment ofpseudouridine excretion by spot urine sampling

valid sampling protocol which would be accu-
rate for a range of diets and feeding systems.

Accuracy of prediction of daily Ps/c
ratios from spot samples

Mean, minimum and maximum correlation co-
efficients between two-hourly Ps/c ratios with
the daily mean (0.558, 0.076 and 0.922, respec-
tively) indicated that single spot samples would
be very unreliable. Assessment of the accuracy
of three sampling regimens showed that accura-
cy of estimation of daily mean Ps/c ratio in-
creased with increasing frequency of spot sam-
pling, an observation which is consistent with
studies evaluating the accuracy of spot sampling
to assess daily mean molar ratios of purine de-
rivatives to creatinine (Daniels et al. 1994, Chen
et al. 1995). The minimum correlation coeffi-
cients demonstrate the worse-case scenarios (Ta-
ble 4). On this basis, even the most intensive
sampling regimen evaluated (four 4-h samples
over two sampling days) would not be reliable
as the r values (between spot sample Ps/c and
daily mean Ps/c determined by total urine col-
lection) could be as low as 0.528 and 0.080 for
treatments SF and CD, respectively.

Relationship between urinary pseudourid-
ine excretion and Ps/c ratios

Prediction of daily pseudouridine excretion from
daily mean Ps/c ratios obtained by a total urine
collection was best achieved by scaling the ra-
tios for live weight (r = 0.700, n = 48, P<0.001).
A similarrelationship has been reported between

daily mean molar ratios of purine derivatives to
creatinine and daily purine derivative excretion
(Daniels et al. 1994). Between-cow variability
in creatinine excretion was mainly responsible
for the poor prediction of pseudouridine excre-
tion indicating that creatinine fails as a marker
of urinary output for individual cows, a finding
which is in agreement with that of Dewhurst
(1989). Current experimental data indicates that
inaccuracies of the spot sampling technique
makes this approach highly unreliable and that
a total urine collection is required to assess ac-
curately urinary pseudouridine excretion for an
individual cow.

Conclusions
There was considerable diurnal variation in the
molar ratio ofpseudouridine to creatinine (Ps/c)
in spot urine samples which was dependent on
feeding system. None of the spot-sampling reg-
imens evaluated gave reliable estimates of the
daily mean Ps/c ratio. Daily mean Ps/c ratios
could account for only half of the variation in
daily urinary pseudouridine excretion largely due
to between-cow variations in creatinine excre-
tion. A total urine collection appears necessary
to assess accurately urinary pseudouridine ex-
cretion in dairy cows.

Acknowledgements. K. J. Shingfield gratefully acknowl-
edges receipt of a studentship provided by the Ministry of
Agriculture, Fisheries and Food, UK. SAC receives finan-
cial support from the Scottish Office Agriculture, Environ-
ment and Fisheries Department. The authors also appreci-
ate the statistical expertise provided by Mr, A. Sword and
Mr. D. Hitchcock.

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Shingfield, K.J. & Offer, N.W. Assessment ofpseudouridine excretion by spot urine sampling

SELOSTUS
Mahdollisuus lyhytaikaisen virtsankeruun käyttöön lypsylehmien

virtsan pseudouridiinin erityksen määrittämisessä
Kevin J. Shingfield jaN.W. Offer

Maatalouden tutkimuskeskus ja SAC Auchincruive, Iso-Britannia

Tutkimuksessa arvioitiin lyhytaikaisen virtsankeruun
soveltuvuutta kokonaispseudouridiinierityksen mää-
ritykseen virtsassa. Kaksitoista useamman kerran poi-
kinutta holstein-friisiläislehmää ruokittiin ristikkäis-
kokeessa kahdella eri koedieetillä kahden 14 päivän
koejakson ajan. Toisella ruokinnalla lehmät saivat
vapaasti säilörehua ja väkirehutäydennys annettiin
kerta-annoksena (SF). Toisella ruokinnalla sama re-
huannos syötettiin seoksena (CD).

Virtsan kokonaiskeräykset tehtiin 24 tunnin ajan
2 tunnin välein koejaksojen päivinä 11 ja 14. Pseu-
douridiinin ja kreatiniinin eritys kullakin 2 tunnin
jaksolla oli yhteydessä keräämisen vuorokaudenai-
kaan (pseudouridiini, P< 0.001 ja kreatiniini,
P<0.05) ja vaihteli lehmittäin (pseudouridiini,
P = 0.092 ja kreatiniini, PcO.Ol), mutta näytteenot-
topäivä tai lehmälle annettu dieetti eivät vaikuttaneet
eritykseen. Pseudouridiinin ja kreatiniinin molaarinen

suhde (Ps/c) vaihteli samalla tavalla vuorokaudenajan
mukaan kuin pseudouridiinin erityskin. Aineistoa
käyttäen määritettiin lyhytaikaisen virtsankeruun täs-
mällisyys Ps/c -suhteen päivittäisen keskiarvon en-
nustamisessa. Useampien näytteiden kerääminen sa-
man päivän aikana osoittautui luotettavammaksi kuin
näytteiden kerääminen useampina päivinä. Lyhytai-
kaisen keruun antaman ennusteen virhe oli suurempi
CD-ruokinnalla kuin SF-dieetillä. Edes kaikkein in-
tensiivisin näytteenotto-ohjelma ei antanut hyväksyt-
täviä ennusteita Ps/c -suhteen päivittäiselle keskiar-
volle, pienimmän r-arvon ollessa 0.528 ja 0.080 SF-
ja CD-ryhmissä. Lisäksi Ps/c -suhteen keskiarvo se-
litti vain puolet havaitusta pseudouridiinin erityksen
päivittäisestä vaihtelusta. Kokonaisvirtsan keräämi-
nen näyttääkin olevan välttämätöntä lypsylehmien
päivittäisen pseudouridiinin erityksen täsmälliseksi
määrittämiseksi.

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AGRICULTURAL AND FOOD SCIENCE IN FINLAND